Dipeptide-catalysed Michael reaction under physiological conditions: Examination of potential bioorthogonality.

Reactions for drug synthesis under cell-like conditions or even inside living cells can potentially be used e.g., to minimize toxic side effects, to maximize bioactive compound efficacy and/or to address drug delivery problems. Those reactions should be bioorthogonal to enable the generation of drug-like compounds with sufficiently good yields. In the known bioorthogonal Michael reactions, using thiols and phosphines as nucleophiles (e.g., in CS and CP bond formation reactions) is very common. No bioorthogonal Michael addition with a carbon nucleophile is known yet. Therefore, the development of such a reaction might be interesting for future drug discovery research. In this work, the metal-free Michael addition between cyclohexanone and various trans-ß-nitrostyrenes (CC bond formation reaction), catalysed by a dipeptide salt H-Pro-Phe-O-Na+, was investigated for the first time in the presence of glutathione (GSH) and in phosphate-buffered saline (PBS). We demonstrated that with electron-withdrawing substituents on the aromatic ring and in ß-position of the trans-ß-nitrostyrene yields up to 64% can be obtained under physiological conditions, indicating a potential bioorthogonality of the studied Michael reaction. In addition, the selected Michael products demonstrated activity against human ovarian cancer cells A2780. This study opens up a new vista for forming bioactive compounds via CC bond formation Michael reactions under physiological (cell-like) conditions.

Batteryless wireless magnetostrictive Fe30Co70/Ni clad plate for human coronavirus 229E detection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been garnered increasing for its rapid worldwide spread. Each country had implemented city-wide lockdowns and immigration regulations to prevent the spread of the infection, resulting in severe economic consequences. Materials and technologies that monitor environmental conditions and wirelessly communicate such information to people are thus gaining considerable attention as a countermeasure. This study investigated the dynamic characteristics of batteryless magnetostrictive alloys for energy harvesting to detect human coronavirus 229E (HCoV-229E). Light and thin magnetostrictive Fe-Co/Ni clad plate with rectification, direct current (DC) voltage storage capacitor, and wireless information transmission circuits were developed for this purpose. The power consumption was reduced by improving the energy storage circuit, and the magnetostrictive clad plate under bending vibration stored a DC voltage of 1.9 V and wirelessly transmitted a signal to a personal computer once every 5 min and 10 s under bias magnetic fields of 0 and 10 mT, respectively. Then, on the clad plate surface, a novel CD13 biorecognition layer was immobilized using a self-assembled monolayer of -COOH groups, thus forming an amide bond with -NH2 groups for the detection of HCoV-229E. A bending vibration test demonstrated the resonance frequency changes because of HCoV-229E binding. The fluorescence signal demonstrated that HCoV-229E could be successfully detected. Thus, because HCoV-229E changed the dynamic characteristics of this plate, the CD13-modified magnetostrictive clad plate could detect HCoV-229E from the interval of wireless communication time. Therefore, a monitoring system that transmits/detects the presence of human coronavirus without batteries will be realized soon.

The effect of acidity on the physicochemical properties of two hydraulic calcium silicate-based cements and two calcium phosphate silicate-based cements.

BACKGROUND: Bioceramic cements have been widely used in endodontic treatment. This study aimed to compare the microhardness, elastic modulus, internal microstructure and chemical compositions of Biodentine, WMTA, ERRM Putty, iRoot FS and IRM after exposure to PBS, butyric acid, and butyric acid followed by PBS. METHODS: Specimens of each material were prepared and randomly divided into 5 subgroups (n = 5): subgroup A: PBS (pH = 7.4) for 4 days, subgroup B: PBS (pH = 7.4) for 14 days, subgroup C: butyric acid (pH = 5.4) for 4 days, subgroup D: butyric acid (pH = 5.4) for 14 days, subgroup E: butyric acid for 4 days followed by 10 days in contact with PBS. The surface microhardness, elastic modulus, internal morphologic and chemical compositions of specimens were analyzed. RESULTS: The microhardness and elastic modulus values of all materials were significantly higher in the presence of PBS compared to exposure to butyric acid, with the same setting time (P < 0.01). After 4-day exposure to butyric acid followed by 10-day exposure to PBS, the microhardness values returned to the same level as 4-day exposure to PBS (P > 0.05). Biodentine showed significantly higher microhardness and elastic modulus values than other materials, while IRM displayed the lowest (P < 0.01). CONCLUSION: Biodentine seems the most suitable bioceramic cements when applied to an infected area with acidic pH. Further storage at neutral pH, e.g. PBS reverses the adverse effects on bioceramic cements caused by a low pH environment.

Anti-inflammatory and immunomodulatory activity of Mangifera indica L. reveals the modulation of COX-2/mPGES-1 axis and Th17/Treg ratio.

In the context of inflammation and immunity, there are fragmented and observational studies relating to the pharmacological activity of Mangifera indica L. and its main active component, mangiferin. Therefore, we aimed to analyze the potential beneficial effects of this plant extract (MIE, 90 % in mangiferin) in a mouse model of gouty arthritis, to allow the evaluation of cellular immune phenotypes and the biochemical mechanism/s beyond MIE activity. Gouty arthritis was induced by the intra-articular administration of MSU crystals (200 µg 20 µl-1), whereas MIE (0.1-10 mg kg-1) or corresponding vehicle (DMSO/saline 1:3) were orally administrated concomitantly with MSU (time 0), 6 and 12 h after the stimulus. Thereafter, knee joint score and oedema were evaluated in addition to western blot analysis for COX-2/mPGES-1 axis. Moreover, the analysis of pro/anti-inflammatory cyto-chemokines coupled with the phenotyping of the cellular infiltrate was performed. Treatment with MIE revealed a dose-dependent reduction in joint inflammatory scores with maximal inhibition observed at 10 mg kg-1. MIE significantly reduced leukocyte infiltration and activation and the expression of different pro-inflammatory cyto-chemokines in inflamed tissues. Furthermore, biochemical analysis revealed that MIE modulated COX-2/mPGES-1 and mPGDS-1/PPARγ pathways. Flow cytometry analysis also highlighted a prominent modulation of inflammatory monocytes (CD11b+/CD115+/LY6Chi), and Treg cells (CD4+/CD25+/FOXP3+) after MIE treatment. Collectively, the results of this study demonstrate a novel function of MIE to positively affect the local and systemic inflammatory/immunological perturbance in the onset and progression of gouty arthritis.

Effects of carrier solutions on the viability and efficacy of canine adipose-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have favorable characteristics that render them a potent therapeutic tool. We tested the characteristics of MSCs after temporal storage in various carrier solutions, such as 0.9% saline (saline), 5% dextrose solution (DS), heparin in saline, and Hartmann's solution, all of which are approved by the U.S. Food and Drug Administration (FDA). Phosphate-buffered saline, which does not have FDA approval, was also used as a carrier solution. We aimed to examine the effects of these solutions on the viability and characteristics of MSCs to evaluate their suitability and efficacy for the storage of canine adipose-derived MSCs (cADMSCs). RESULTS: We stored the cADMSCs in the test carrier solutions in a time-dependent manner (1, 6, and 12 h) at 4 °C, and analyzed cell confluency, viability, proliferation, self-renewability, and chondrogenic differentiation. Cell confluency was significantly higher in 5% DS and lower in phosphate-buffered saline at 12 h compared to other solutions. cADMSCs stored in saline for 12 h showed the highest viability rate. However, at 12 h, the proliferation rate of cADMSCs was significantly higher after storage in 5% DS and significantly lower after storage in saline, compared to the other solutions. cADMSCs stored in heparin in saline showed superior chondrogenic capacities at 12 h compared to other carrier solutions. The expression levels of the stemness markers, Nanog and Sox2, as well as those of the MSC surface markers, CD90 and CD105, were also affected over time. CONCLUSION: Our results suggest that MSCs should be stored in saline, 5% DS, heparin in saline, or Hartmann's solution at 4 °C, all of which have FDA approval (preferable storage conditions: less than 6 h and no longer than 12 h), rather than storing them in phosphate-buffered saline to ensure high viability and efficacy.

Prospects of NIR fluorescent nanosensors for green detection of SARS-CoV-2.

The pandemic of the novel coronavirus disease 2019 (COVID-19) is continuously causing hazards for the world. Effective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can relieve the impact, but various toxic chemicals are also released into the environment. Fluorescence sensors offer a facile analytical strategy. During fluorescence sensing, biological samples such as tissues and body fluids have autofluorescence, giving false-positive/negative results because of the interferences. Fluorescence near-infrared (NIR) nanosensors can be designed from low-toxic materials with insignificant background signals. Although this research is still in its infancy, further developments in this field have the potential for sustainable detection of SARS-CoV-2. Herein, we summarize the reported NIR fluorescent nanosensors with the potential to detect SARS-CoV-2. The green synthesis of NIR fluorescent nanomaterials, environmentally compatible sensing strategies, and possible methods to reduce the testing frequencies are discussed. Further optimization strategies for developing NIR fluorescent nanosensors to facilitate greener diagnostics of SARS-CoV-2 for pandemic control are proposed.

Perindopril sensitizes hepatocellular carcinoma to chemotherapy: A possible role of leptin / Wnt/ ß-catenin axis with subsequent inhibition of liver cancer stem cells.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death. The major challenge in managing HCC is the resistance to chemotherapy. Leptin hormone is associated with different oncogenic pathways implicated in drug resistance. Angiotensin II was found to decrease the production and secretion of leptin. Objective: This study investigated the potential role of an ACEI perindopril as a chemosensitizer agent to sorafenib. Method: HCC was induced in mice using a single dose of diethylnitrosamine DENA (200 mg/kg) followed by phenobarbital 0.05% in drinking water for 16 weeks. Mice were then treated with perindopril (1 mg/kg/day), Sorafenib (30 mg/kg/day), or both of them for another four weeks. Leptin, VEGF, MMP-9, Cyclin D1, EpCAM, and ß-catenin were measured using immunoassay while Wnt and ALDH1 were assayed using western blotting assay. Results: Perindopril whether alone or in combination with sorafenib decrease liver enzymes and preserve the liver architecture. Our study revealed that perindopril significantly increased the antineoplastic, antiangiogenic as well as anti-metastatic effects of sorafenib. This effect was correlated with the downregulation of the leptin / Wnt / ß-catenin pathway and overexpression of ALDH1 while downregulation of EpCAM. Conclusion: This study presents perindopril as a potential chemosensitizer agent that works through decreased expression of the leptin / Wnt / ß-catenin pathway.

Genistein promotes M1 macrophage apoptosis and reduces inflammatory response by disrupting miR-21/TIPE2 pathway.

Cardiovascular diseases are a major cause of mortality, and vascular injury, a common pathological basis of cardiovascular disease, is deeply correlated with macrophage apoptosis and inflammatory response. Genistein, a type of phytoestrogen, exerts cardiovascular protective activities, but the underlying mechanism has not been fully elucidated. In this study, RAW264.7 cells were treated with genistein, lipopolysaccharide (LPS), nuclear factor-kappa B (NF-κB) inhibitor, and/or protein kinase B (AKT) agonist to determine the role of genistein in apoptosis and inflammation in LPS-stimulated cells. Simultaneously, high fat diet-fed C57BL/6 mice were administered genistein to evaluate the function of genistein on LPS-induced cardiovascular injury mouse model. Here, we demonstrated that LPS obviously increased apoptosis resistance and inflammatory response of macrophages by promoting miR-21 expression, and miR-21 downregulated tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) expression by targeting the coding region. Genistein reduced miR-21 expression by inhibiting NF-κB, then blocked toll-like receptor 4 (TLR4) pathway and AKT phosphorylation dependent on TIPE2, resulting in inhibition of LPS. Our research suggests that miR-21/TIPE2 pathway is involved in M1 macrophage apoptosis and inflammatory response, and genistein inhibits the progression of LPS-induced cardiovascular injury at the epigenetic level via regulating the promoter region of Vmp1 by NF-κB.

Fracture resistance of human roots filled with mineral trioxide aggregate mixed with phosphate-buffered saline, with and without calcium hydroxide pre-medication.

AIM: To compare the fracture strength of extracted human roots with apical plugs of mineral trioxide aggregate (MTA) mixed with either Ca- and Mg-free phosphate-buffered saline (PBS) or water, with and without calcium hydroxide (CH) canal pre-medication. METHODOLOGY: A total of 180 single-rooted human teeth were prepared to resemble immature roots and divided into groups (n = 20). The negative control received canal irrigation only, and the positive control received intracanal treatment with CH for either two or twelve weeks. MTA mixed with water was used in Group 1: (i) without CH pre-medication - MTA(W); (ii) after 2 weeks CH pre-medication - 2/52CH + MTA(W); and (iii) after 12-week CH pre-medication - 12/52 CH + MTA(W). MTA mixed with PBS was used in Group 2: (i) without CH pre-medication - MTA(PBS); (ii) after 2-week CH pre-medication - 2/52CH + MTA(PBS); and (iii) after 12-week CH pre-medication - 12/52 CH + MTA(PBS). A compressive force was applied to each root until the point of fracture. The results were analysed by the Kruskal-Wallis and Dunn's multiple comparisons tests (P < 0.05). RESULTS: There was no significant difference between groups MTA(W), MTA(PBS) and 2/52CH + MTA(PBS), and all three groups were significantly (P < 0.01, P < 0.05 and P < 0.05, respectively) more resistant to fracture than the negative control. Within Group 1, the samples that received two- (P < 0.01) and twelve-week (P < 0.001) CH pre-treatment were more prone to fracture than those which did not. No difference was found amongst the control groups. The roots of the MTA(PBS) group had a higher dependability (P < 0.05) than the MTA(W) group when compared by the Weibull modulus. The difference was also present when a 2-week CH pre-medication was used. CONCLUSIONS: Mineral trioxide aggregate mixed with Ca- and Mg-free phosphate-buffered saline had a significant strengthening effect on the fracture resistance of structurally weak roots, even when short-term calcium hydroxide pre-medication had been used. MTA mixed with water lost its strengthening effect on human roots when 2- or 12-week CH pre-treatment had been used. Use of CH dressing for up to 12 weeks had no negative effect on fracture resistance of human roots.

Commiphora myrrha (Nees) Engl. resin extracts induce phase-I cytochrome P450 2C8, 2C9, 2C19, and 3A4 isoenzyme expressions in human hepatocellular carcinoma (HepG2) cells.

Commiphora myrrha (Nees) Engl. (C. myrrha) resin is the most Middle Eastern herbal medicine used against numerous diseases. After being decocted or macerated, this resin is widely consumed among Saudi Arabian patients who are already under prescribed medication. Despite its popularity, no studies have been reported on potential modulation effects of these resin extracts on drug metabolism. Therefore, we studied C. myrrha resin extracts on the expression of cytochrome P450 (CYP) drug-metabolizing isoenzyme in human hepatocellular carcinoma cell line HepG2. The C. myrrha extracts were prepared by sonication and boiling, resembling the most popular traditional preparations of maceration and decoction, respectively. Both boiled and sonicated aqueous extracts were fingerprinted using high-performance liquid chromatography equipped with ultra-violet detector (HPLC-UVD). The viability of HepG2 cells treated with these aqueous extracts was determined using CellTiter-Glo® assay in order to select the efficient and non-toxic resin extract concentrations for phase-I metabolic CYP isoenzyme expression analysis. The isoenzyme gene and protein expression levels of CYP 2C8, 2C9, 2C19, and 3A4 were assessed using reverse transcription-quantitative polymerase chain reaction and Western blot technologies. The HPLC-UVD fingerprinting revealed different chromatograms for C. myrrha boiled and sonicated aqueous extracts. Both aqueous extracts were toxic to HepG2 cells when tested at concentrations exceeding 150 µg/ml of the dry crude extract. The CYP 2C8, 2C9, and 2C19 mRNA expression levels increased up to 4.0-fold in HepG2 cells treated with either boiled or sonicated C. myrrha aqueous extracts tested between 1 and 30 µg/ml, as compared with the untreated cells. However, CYP3A4 mRNA expression level exceeded the 2.0-fold cutoff when the cells were exposed to 30 µg/ml of C. myrrha extracts. The up-regulation of CYP mRNA expression levels induced by both boiled and sonicated C. myrrha aqueous extracts was confirmed at the CYP protein expression levels. In conclusion, both sonicated and boiled C. myrrha aqueous extracts modulate CYP 2C8, 2C9, 2C19, and 3A4 gene expression at clinically-relevant concentrations regardless of preparation methods. Further in vitro and in vivo experiments are required for CYP isoenzyme activity assessment and the establishment of herb-drug interaction profile for these traditional medicinal resin extracts.

Cinnamomum zeylanicum Blume (Ceylon cinnamon) bark extract attenuates doxorubicin induced cardiotoxicity in Wistar rats.

Anti-tumour efficacy of doxorubicin is hindered by the cumulative dose-dependent cardiotoxicity induced by reactive oxygen species during its metabolism. As Cinnamomum zeylanicum has proven antioxidant potential, objective of this study was to investigate the cardioprotective activity of Cinnamomum bark extract against doxorubicin induced cardiotoxicity in Wistar rats. Physicochemical and phytochemical analysis was carried out and dose response effect and the cardioprotective activity of Cinnamomum were determined in vivo. 180 mg/kg dexrazoxane was used as the positive control. Plant extracts were free of heavy metals and toxic phytoconstituents. In vivo study carried out in Wistar rats revealed a significant increase (p < 0.05) in cardiac troponin I, NT-pro brain natriuretic peptide, AST and LDH concentrations in the doxorubicin control group (18 mg/kg) compared to the normal control. Rats pre-treated with the optimum dosage of Cinnmamomum (2.0 g/kg) showed a significant reduction (p < 0.05) in all above parameters compared to the doxorubicin control. A significant reduction was observed in the total antioxidant capacity, reduced glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase activity while the lipid peroxidation and myeloperoxidase activity were significantly increased in the doxorubicin control group compared to the normal control (p < 0.05). Pre-treatment with Cinnamomum bark showed a significant decrease in lipid peroxidation, myeloperoxidase activity and significant increase in rest of the parameters compared to the doxorubicin control (p < 0.05). Histopathological analysis revealed a preserved appearance of the myocardium and lesser degree of cellular changes of necrosis in rats pre-treated with Cinnamomum extract. In conclusion, Cinnamomum bark extract has the potential to significantly reduce doxorubicin induced oxidative stress and inflammation in Wistar rats.

Immersion of Achilles tendon in phosphate-buffered saline influences T1 and T2 * relaxation times: An ex vivo study.

Robust mapping of relaxation parameters in ex vivo tissues is based on hydration and therefore requires control of the tissue treatment to ensure tissue integrity and consistent measurement conditions over long periods of time. One way to maintain the hydration of ex vivo tendon tissue is to immerse the samples in a buffer solution. To this end, various buffer solutions have been proposed; however, many appear to influence the tissue relaxation times, especially with prolonged exposure. In this work, ovine Achilles tendon tissue was used as a model to investigate the effect of immersion in phosphate-buffered saline (PBS) and the effects on the T1 and T2* relaxation times. Ex vivo samples were measured at 0 (baseline), 30 and 67 hours after immersion in PBS. Ultrashort echo time (UTE) imaging was performed using variable flip angle and echo train-shifted multi-echo imaging for T1 and T2* estimation, respectively. Compared with baseline, both T1 and T2* relaxation time constants increased significantly after 30 hours of immersion. T2* continued to show a significant increase between 30 and 67 hours. Both T1 and T2* tended to approach saturation at 67 hours. These results exemplify the relevance of stringently controlled tissue preparation and preservation techniques, both before and during MRI experiments.

Endogenous Fluorescence Dissimilarity Assessment of Four Potential Biomarkers of Early Liver Fibrosis by Preservation Media Effect.

Build-up of extracellular matrix in liver fibrosis results in changes on endogenous molecules expression that may be studied through the fluorescence characterization of ex vivo liver samples. To the best of our knowledge, no investigations have provided in-depth evidence and discussion on the changes of the endogenous fluorescence in ex vivo tissue due to the effects of the preservation media. In this work, we contrast and analyze the endogenous fluorescence from tryptophan, vitamin A, hydroxyproline and elastin cross-links potential biomarkers of the liver fibrosis, in in vivo measurements and liver samples preserved on formaldehyde, and two standard preservation media. As it is known, chemical changes in tissue, caused by formaldehyde fixation, alter the endogenous fluorescence spectra. We propose the use of phosphate-buffered saline (PBS), and Iscove's Modified Dulbecco's Medium (IMDM) to elude the fluorescence changes. PBS and IMDM showed to maintain the endogenous fluorescence characteristics similar to in vivo conditions. The results of this work point the way for a more reliable assessment of endogenous fluorescence in ex vivo hepatic studies.

Fraxetin inhibits interleukin-1ß-induced apoptosis, inflammation, and matrix degradation in chondrocytes and protects rat cartilage in vivo.

Osteoarthritis (OA) is a disease characterized by degeneration of the joint complex due to cartilage destruction. Fraxetin, a widely used and studied coumarin compound extracted from a traditional Chinese herb (Qin Pi), has shown anti-inflammatory and antioxidant properties, but its effects on OA have not been studied. In the present study, western blotting, immunofluorescence, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) were used to evaluate the effects of fraxetin on IL-1ß-induced apoptotic activity, inflammatory responses, and catabolism in rat chondrocytes. The results showed that fraxetin prevented IL-1ß-induced apoptosis of chondrocytes and inhibited inflammatory mediator release by regulating the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor (NF)-κB pathway in chondrocytes. Additionally, fraxetin suppressed the upregulation of matrix metalloproteinase 13 (MMP13) and degradation of collagen II in the extracellular matrix (ECM). Moreover, the effects of fraxetin in vivo were assessed in a monosodium iodoacetate (MIA)-induced rat model of OA using hematoxylin and eosin (H&E) and Safranin O-fast green staining and magnetic resonance imaging (MRI). The results showed that fraxetin protected the cartilage against destruction. In conclusion, fraxetin could be a potential therapeutic for OA.

Codium fragile F2 sensitize colorectal cancer cells to TRAIL-induced apoptosis via c-FLIP ubiquitination.

This study demonstrates that combined treatment with subtoxic doses of Codium extracts (CE), a flavonoid found in many fruits and vegetables, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induces apoptosis in TRAIL-resistant colorectal cancer (CRC) cells. Effective induction of apoptosis by combined treatment with CE and TRAIL was not blocked by Bcl-xL overexpression, which is known to confer resistance to various chemotherapeutic agents. While TRAIL-mediated proteolytic processing of procaspase-3 was partially blocked in various CRC cells treated with TRAIL alone, co-treatment with CE efficiently recovered TRAIL-induced caspase activation. We observed that CE treatment of CRC cells did not change the expression of anti-apoptotic proteins and pro-apoptotic proteins, including death receptors (DR4 and DR5). However, CE treatment markedly reduced the protein level of the short form of the cellular FLICE-inhibitory protein (c-FLIPS), an inhibitor of caspase-8, via proteasome-mediated degradation. Collectively, these observations show that CE recovers TRAIL sensitivity in various CRC cells via down-regulation of c-FLIPS.

Epithelial cells exert differential traction stress in response to substrate stiffness.

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of studies, the effects that environmental changes have on this form of movement are poorly understood. To examine the role of substrate compliancy on multi-layered epithelial sheet migration, we performed traction force and confocal microscopy to determine differences in traction forces and to examine focal adhesions on synthetic and biological substrates. The leading edges of corneal epithelial sheets undergo retraction or contraction prior to migration, and alterations in the sheet's stiffness are affected by the amount of force exerted by cells at the leading edge. On substrates of 30 kPa, cells exhibited greater and more rapid movement than on substrates of 8 kPa, which are similar to that of the corneal basement membrane. Vinculin and its phosphorylated residue Y1065 were prominent along the basal surface of migrating cells, while Y822 was prominent between neighboring cells along the leading edge. Vinculin localization was diffuse on a substrate where the basement membrane was removed. Furthermore, when cells were cultured on fibronectin-coated acrylamide substrates of 8 and 50 kPa and then wounded, there was an injury-induced phosphorylation of Y1065 and substrate dependent changes in the number and size of vinculin containing focal adhesions. These results demonstrate that changes in substrate stiffness affected traction forces and vinculin dynamics, which potentially could contribute to the delayed healing response associated with certain corneal pathologies.

IL-17A neutralizing antibody regulates monosodium urate crystal-induced gouty inflammation.

Gout is a paradigm of acute, self-limiting inflammation caused by the deposition of monosodium urate (MSU) crystals within intra-and/or peri-articular areas, leading to excruciating pain, joint swelling and stiffness. The infiltration of leukocytes drives the inflammatory response and remains an attractive target for therapeutic intervention. In this context, emerging evidence supports the view that systemic differentiation of Th17 cells and their in situ infiltration as one of the potential mechanisms by which these cells, and their main product IL-17, causes damage to target tissues. To test if IL-17 was having a detrimental role in gouty onset and progression we targeted this cytokine, using a neutralizing antibody strategy, in an experimental model of gout. Joint inflammation was induced in CD-1 mice by the intra-articular (i.a.) administration of MSU crystals (200 µg/20 µl). Animals from IL-17Ab-treated groups received 1, 3 and 10 µg (i.a.) in 20 µl of neutralizing antibody after MSU crystals administration. Thereafter, joints were scored macroscopically, and knee joint oedema determined with a caliper. Histological analysis, myeloperoxidase assay and western blots analysis for COX-2/mPGEs-1/IL-17R pathway were conducted at 18 h (peak of inflammation) to evaluate leukocytes infiltration and activation, followed by the analysis, in situ, of pro/anti-inflammatory cytokines and chemokines. Flow cytometry was also used to evaluate the modulation of infiltrated inflammatory monocytes and systemic Th17 and Treg profile. Treatment with IL-17Ab revealed a dose-dependent reduction of joint inflammation scores with maximal inhibition at 10 µg. The neutralizing antibody was also able to significantly reduce leukocytes infiltration and MPO activity as well the expression of JE, IL-1α, IL-1ß, IL-16, IL-17, C5a, BLC and, with a less extent IP-10, Rantes, KC, TIMP-1, SDF-1 and metalloproteinases in inflamed tissues. Biochemical analysis also revealed that IL-17Ab treatment modulated COX-2/mPGEs-1 pathway (and related PGE2 production) without interfering with IL-17R expression. Furthermore, flow cytometry analysis highlighted a selective modulation of infiltrating inflammatory monocytes (B220-/GR1hi-F480hi/CD115+) and circulating Th17, but not Treg, cells after IL-17Ab treatment. Collectively the results of this study report for the first time, that i.a. injection of MSU crystals stimulates in vivo production of Th17 cells and Th17-related inflammatory cyto-chemokines. In addition, we have demonstrated that the administration of a neutralizing antibody against IL-17 attenuates joint symptoms, swelling and leukocytes infiltration to the inflamed tissue, possibly providing a new strategy for the treatment of gouty inflammation and/or arthritis.

Characterization of Phosphate Buffered Saline (PBS) in Frozen State and after Freeze-Drying.

PURPOSE: To study the effect of mannitol or trehalose on the crystallization behavior of solutes in phosphate buffered saline (PBS) when the solutions were frozen and freeze-dried. METHODS: PBS (pH 7.5 at RT) either alone, or with trehalose (5% w/v) or mannitol (1% w/v), were frozen and characterized using low temperature differential scanning calorimetry (DSC), X-ray diffractometry (XRD), and pH measurement. Freeze dried lyophiles were characterized by XRD. RESULTS: In the absence of cosolutes, upon freezing PBS, a pH shift of ~ 4 units was observed due to crystallization of Na2HPO4•12H2O. XRD indicated sequential crystallization of Na2HPO4•12H2O, NaCl•2H2O and KCl during cooling. When the frozen solutions were heated, two eutectics were observed - the first at ~ -24°C (ternary, NaCl•2H2O-KCl-ice) and the second at ~ -22°C (binary, NaCl•2H2O-ice). Trehalose completely inhibited buffer salt crystallization, whereas mannitol suppressed it partially thereby attenuating the magnitude of pH shift. The two eutectic meltings were also suppressed by the cosolutes. XRD of final lyophiles from PBS alone revealed peaks of anhydrous Na2HPO4, NaCl, and KCl. Trehalose rendered the lyophiles completely XRD amorphous, whereas in presence of mannitol, all the solutes except KH2PO4 crystallized. CONCLUSIONS: Freezing of PBS solution caused a pronounced pH shift due to selective crystallization of Na2HPO4•12H2O. The addition of trehalose or mannitol suppressed the buffer salt crystallization and attenuated the magnitude of pH shift. The potential instability of biologics due to pH shift in PBS, can be potentially mitigated with the cosolutes.

Development and Utilization of VHH Antibodies Derived from Camelus Dromedarius Against Foot-and-Mouth Disease Virus.

Foot-and-mouth disease (FMD) is an acute, highly contagious, and economically devastating viral disease of domestic and wildlife species. For effective implementation of FMD control program, there is an imperative need for developing a rapid, sensitive, and specific diagnostics which help in the identification of serotypes involved in the outbreaks. The humoral immune response of the Camelidae is unique since in these animals 75% of circulating antibodies are constituted by heavy-chain antibodies and 25% are conventional immunoglobulin with two identical heavy chains. In the present study, we developed and characterized FMD virus-specific single-domain heavy-chain antibodies (VHHs) against inactivated whole-virus antigens of FMDV serotypes O (INDR2/1975), A (IND40/2000), and Asia 1 (IND63/1972) vaccine strains. After six rounds of panning and enrichment, these VHHs were stably expressed in Escherichia coli cells. The VHHs directed against outer capsid proteins of FMD virus were successfully utilized as the capture antibody in liquid-phase blocking ELISA (LPBE) thus replacing rabbit coating antibodies. Our study demonstrated the utility of FMD virus-specific VHHs as potential candidates in FMD research and diagnostic application.

Expression and intracellular localization of Nanos2-homologue protein in primordial germ cells and spermatogonial stem cells.

SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.